Drug delivery system comprising a tetrahydroxylated estrogen for use in hormonal contraception

ABSTRACT

A method of contraception in mammalian females, which method comprises the parenteral or rectal administration of an estrogenic component and a progestogenic component to a female of childbearing capability in an amount effective to inhibit ovulation, wherein the estrogenic component is selected from the group consisting of substances represented by the following formula (1) 
                         
in which R 1 , R 2 , R 3 , R 4  independently are a hydrogen atom, a hydroxyl group or an alkoxy group with 1-5 carbon atoms; each of R 5 , R 6 , R 7  is a hydroxyl group; and no more than 3 of R 1 , R 2 , R 3 , R 4  are hydrogen atoms; precursors capable of liberating a substance according to the aforementioned formula when used in the present method; and mixtures of one or more of the aforementioned substances and/or precursors. Another aspect of the invention concerns a drug delivery system for parenteral or rectal administration that contains the aforementioned estrogenic component and a progestogenic component, said drug delivery system being selected from the group consisting of suppositories, systems for intravaginal delivery, inhalers, nasal sprays and transdermal delivery systems.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a method of hormonal contraception inmammalian females. More particularly the invention is concerned with amethod of hormonal contraception that comprises the parenteral or rectaladministration of a combination of an estrogenic component and aprogestogenic component to a female of childbearing capability in aneffective amount to inhibit ovulation.

The invention also encompasses a pharmaceutical kit comprising theaforementioned estrogenic component and a progestogenic component.

BACKGROUND OF THE INVENTION

Estrogens play an important major role in existing methods of hormonalcontraception. For contraception estrogens are commonly used togetherwith a progestogen, e.g. levonorgestrel, desogestrel, norethisterone,cyproterone acetate, dienogest. The estrogens are needed for inhibitingfollicle maturation and ovulation, but in addition they replace theendogenous ovarian secretion of estradiol which is suppressed to a majorextent by the administration of a hormonal contraceptive. Thisreplacement is important for preventing estrogen deficiency and formaintaining an artificial menstrual cycle and other genital functions.

Endogenous and exogenous estrogens fulfil important central nervous andmetabolic functions in the female organism: normal estrogen levels makea decisive contribution to a woman's well-being. Notwithstanding thewidespread use of estrogens in hormonal contraceptives, there are stillsome unsolved problems. Known estrogens, in particular the biogenicestrogens (i.e. estrogens that occur naturally in the human body), areeliminated from the blood stream very quickly. For instance, for themain human biogenic estrogen 17β-estradiol the half-life is around 1hour. As a result, between separate administration events, blood serumlevels of such biogenic estrogens tend to fluctuate considerably. Thus,shortly after administration the serum concentration is usually severaltimes higher than the optimum concentration. In addition, if the nextadministration event is delayed, serum concentrations will quicklydecrease to a level where the estrogen is no longer physiologicallyactive. This is particularly undesirable in contraceptive methods.

The most important synthetically altered estrogenic steroid is17α-ethinyl estradiol (EE). This estrogen is dominant in oral hormonalcontraception. Apart from EE, mestranol has been used in a few cases;mestranol is a “prodrug” that is metabolised to EE in the organism. Theliver is a target organ for estrogens. The secretion activity that isaffected by estrogens in the human liver includes increased synthesis oftransport proteins CBG, SHBG, TBG, several factors that are importantfor the physiology of blood clotting, and lipoproteins. The stronghepatic estrogenicity of ethinyl estradiol and diethylstilbestrol (DES),especially their effects on haemostasis factors, may explain why thesesynthetic estrogens have been associated with the enhanced risk ofthromboembolism. Other undesirable side-effects that have been reportedin relation to the use of synthetic estrogens include fluid retention,nausea, bloating, cholelithiasis, headache and breast pain.

The aforementioned deficits are of considerable clinical significancewhen commonly known biogenic or synthetic estrogens are applied.Consequently, there is an as yet unmet need for estrogens that do notdisplay these deficits and which can suitably be employed incontraceptive methods for females because of their ability to (a)reliably suppress follicle maturation and ovulation and to (b)effectively replace the endogenous ovarian secretion of 17β-estradiol.

SUMMARY OF THE INVENTION

The inventors have surprisingly found that these objectives are met byestrogenic substances that are represented by the following formula

in which formula R₁, R₂, R₃, R₄ independently are a hydrogen atom, ahydroxyl group or an alkoxy group with 1-5 carbon atoms; each of R₅, R₆,R7 is a hydroxyl group; and no more than 3 of R₁, R₂, R₃, R₄ arehydrogen atoms.

A known representative of this group of estrogenic substances is 1,3,5(10)-estratrien-3, 15α,16α,17β-tetrol, also known by the names ofestetrol, oestetrol and 15α-hydroxyestriol. Estetrol is an estrogen thatis produced by the fetal liver during human pregnancy. Unconjugatedestetrol levels in maternal plasma peak at about 1.2 ng/ml at termpregnancy and are about 12 times higher in fetal than in maternal plasma(Tulchinsky et. al., 1975. J. Clin. Endocrinol. Metab., 40, 560-567).

In 1970,Fishman et al., “Fate of 15α-hydroxyestriol-³H in Adult Man”, JClin Endocrinol Metab (1970) 31, 436-438,reported the results of a studywherein tritium labeled 15α-hydroxyestriol (estetrol) was administeredintravenously to two adult women. It was found that the estetrol wasrapidly and completely excreted in urine as the glucosiduronate and thatvirtually no metabolism except for conjugation took place.

Between 1975 and 1985 several researchers have investigated theproperties of estetrol and reported on its estrogenic potency anduterotrophic activity. The most relevant publications that were issuedduring this period are mentioned below:

-   -   Levine et al., 1984. Uterine vascular effects of estetrol in        nonpregnant ewes. Am. J. Obstet. Gynecol., 148:73, 735-738:        “When intravenously administered in nonpregnant ewes, estetrol        is 15 to 30 times less potent than estriol and 17β-estradiol in        uterine vasodilation”.    -   Jozan et al., 1981. Different effects of oestradiol, oestriol,        oestetrol and of oestrone on human breast cancer cells (MCF-7)        in long term-tissue culture. Acta Endocrinologica, 98, 73-80:        “Estetrol agonistic potency is 2% of the magnitude observed for        17β-estradiol in in vitro cell proliferation”.    -   Holinka et al., 1980. Comparison of effects of estetrol and        tamoxifen with those of estriol and estradiol on the immature        rat uterus. Biol. Reprod. 22, 913-926: “Subcutaneously        administered estetrol has very weak uterotrophic activity and is        considerable less potent than 17β-estradiol and estriol”.    -   Holinka et al., 1979. In vivo effects of estetrol on the        immature rat uterus. Biol. Reprod. 20, 242-246: “Subcutaneously        administered estetrol has very weak uterotrophic activity and is        considerable less potent than 17β-estradiol and estriol”.    -   Tseng et al., 1978. Heterogeneity of saturable estradiol binding        sites in nuclei of human endometrium. Estetrol studies. J.        Steroid Biochem. 9, 1145-1148: “Relative binding of estetrol to        estrogen receptors in the human endometrium is 1.5% of        17β-estradiol”.    -   Martucci et al., 1977. Direction of estradiol metabolism as a        control of its hormonal action-uterotrophic activity of        estradiol metabolites. Endocrin. 101, 1709-1715: “Continuous        administration of estetrol from a subcutaneous depot shows very        weak uterotrophic activity and is considerably less potent than        17β-estradiol and estriol”.    -   Tseng et al., 1976. Competition of estetrol and ethynylestradiol        with estradiol for nuclear binding in human endometrium. J.        Steroid Biochem. 7, 817-822: “The relative binding constant of        estetrol binding to the estrogen receptor in the human        endometrium is 6.25% compared to 17β-estradiol (100%)”.    -   Martucci et al., 1976. Uterine estrogen receptor binding of        catecholestrogens and of estetrol        (1,3,5(10)-estratriene-3,15alpha,16alpha, 17beta-tetrol).        Steroids, 27, 325-333: “Relative binding affinity of estetrol to        rat uterine cytosol estrogen receptor is 0.5% of 17β-estradiol        (100%). Furthermore, the relative binding affinity of estetrol        to rat uterine nuclear estrogen receptor is 0.3% of        17β-estradiol (100%)”.

All of the above publications have in common that the authors haveinvestigated the estrogenic potency of estetrol. Without exception theyall conclude that estetrol is a weak estrogen. In some of the citedarticles the estrogenic potency of estetrol has been found to be lowerthan that of another biogenic estrogen, namely, 17β-estradiol, which isconsidered to be a relatively weak estrogen (e.g. compared to ethinylestradiol). With these findings in mind, it is not surprising that theinterest in estetrol has dwindled since the early eighties and that nopublications on the properties of estetrol have been issued since.

U.S. Pat. No. 5,468,736 (Hodgen) describes a method of hormonereplacement therapy involving the administration of estrogen togetherwith an amount of antiprogestin (antiprogestogen), which inhibitsestrogen-induced endometrial proliferation in women. In Example 3 thecombined use of estetrol and lilopristone is mentioned. No clues aregiven in the examples as to the mode and frequency of administration orregarding the dosage level employed. A disadvantage associated with theuse of antiprogestogens, such as lilopristone, is the risk of inducingabnormal endometrial morphology, i.e. cystic hyperplasia, as has beenobserved in women who received an antiprogestogen treatment againstendometriosis (Murphy et al., 1995. Fertil. Steril., 95, 761-766).

U.S. Pat. No. 5,340,586 (Pike et al.) is concerned with compositions andmethods which are effective to treat oophorectomised women, wherein aneffective amount of an estrogenic composition and an androgeniccomposition are provided over a period of time. In the US-patent it isstated that natural and synthetic estrogenic compositions that can beused include natural estrogenic hormones and congeners, including butnot limited to estradiol, estradiol benzoate, estradiol cypionate,estradiol valerate, estrone, diethylstilbestrol, piperazine estronesulfate, ethinyl estradiol, mestranol, polyestradiol phosphate, estriol,estriol hemisuccinate, quinestrol, estropipate, pinestrol and estronepotassium sulfate, and furthermore that equine estrogens, such asequilelinin, equilelinin sulfate and estetrol, may also be employed.Except for the exhaustive inventory of known estrogens, no otherreference to estetrol (which is erroneously referred to as an equineestrogen) is made in this US-patent.

The same exhaustive list of estrogens is found in the following patentdocuments:

-   -   U.S. Pat. No. 4,762,717 (Crowley): A contraceptive method        comprising the sequential administration of (1) a combination of        luteinizing hormone releasing hormone (LHRH) and estrogen        and (2) a combination of LHRH and estrogen and progestogen.    -   U.S. Pat. No. 5,130,137 (Crowley): A method of treating benign        ovarian secretory disorder comprising the sequential        administration of (1) a combination of luteinizing hormone        releasing hormone (LHRH) and estrogen and (2) a combination of        LHRH and estrogen and progestogen.    -   U.S. Pat. No. 5,211,952 (Spicer et al.): A contraceptive method        comprising administering a gonadotropin hormone releasing        hormone (GnRH) composition in an amount effective to inhibit        ovulation and administering estrogen and progestogen to maintain        serum levels above a defined minimum level.

U.S. Pat. No. 5,340,584 (Spicer et al.): A method for preventingconception or for treating benign gynaecological disorders comprisingadministering a GnRH composition for a first period of time in an amounteffective to suppress ovarian estrogen and progesterone production,simultaneously administering an estrogenic composition in an amounteffective to prevent symptoms of estrogen deficiency and simultaneouslyadministering a progestogen in an amount effective to maintain serumlevel of said progestogen at a level effective to decrease endometrialcell proliferation.

-   -   U.S. Pat. No. 5,340,585 (Pike et al.): A method of treating        benign gynaecological disorders in a patient in whom the risk of        endometrial stimulation by estrogenic compositions is minimised        or absent, comprising administering a GnRH composition in an        amount effective to suppress ovarian estrogen and progesterone        production and administering an estrogenic composition in an        amount effective to prevent symptoms of estrogen deficiency.    -   WO 00/73416 (Yifang et al.): A method for regulating the        fertility of a host, comprising contacting host ovarian cells        with a safe and effective amount of a pharmaceutical composition        comprising an antisense oligonucleotide that is complementary to        the nucleotide sequence of the follicle stimulating hormone        (FSH) receptor. The possibility of combined administration of        such an antisense oligonucleotide with an estrogenic steroid is        mentioned in the application.

The benefits of the present invention may be realised without theco-administration of anti-progestogens, LHRH compositions, GnRHcompositions and/or antisense oligonucleotides that are complementary tothe nucleotide sequence of the follicle stimulating hormone (FSH)receptor as proposed in the aforementioned publications. Also, thepresent invention may suitably be applied in individuals who have notbeen oophorectomised, or in whom the risk of endometrial stimulation byestrogenic compositions is not minimised or absent, other than bycombined administration of a progestogen and an estrogen, e.g. as aresult of hysterectomy. Furthermore the present method does not requirethe use of a slow release formulation as is dictated by most of theaforementioned US-patents.

In view of the low estrogenic potency of the estetrol-like substancesthat are employed in accordance with the invention, it is surprisingthat these substances can effectively be used in a contraceptive method.Although the inventors do not wish to be bound by theory, it is believedthat the unexpected efficacy of parenterally or rectally administeredestetrol-like substances results from the combination of unforeseenfavourable pharmacokinetic (ADME) and pharmacodynamic properties ofthese substances.

As regards the pharmacokinetic properties of the present estrogenicsubstances the inventors have discovered that their in vivo half-life isconsiderably longer than that of other biogenic estrogens. Thus, eventhough estetrol and estetrol-like substances have relatively lowestrogenic potency, they may effectively be employed in a contraceptivemethod because their low potency is compensated for by a relatively highmetabolic stability, as demonstrated by a long half-life.

An advantageous property of the present estrogenic substances resides inthe fact that sex hormnone-binding globulin (SHBG) hardly binds theseestrogenic substances, meaning that, in contrast to most knownestrogens, serum levels are representative for bio-activity andindependent of SHBG levels.

Another important benefit of the present estrogenic substances isderived from their relative insensitivity to interactions with otherdrugs (drug-drug interactions). It is well known that certain drugs maydecrease the effectiveness of estrogens, such as ethinyl estradiol, andother drugs may enhance their activity, resulting in possible increasedside-effects. Similarly estrogens may interfere with the metabolism ofother drugs. In general, the effect of other drugs on estrogens is dueto interference with the absorption, metabolism or excretion of theseestrogens, whereas the effect of estrogens on other drugs is due tocompetition for metabolic pathways.

The clinically most significant group of estrogen-drug interactionsoccurs with drugs that may induce hepatic microsomal enzymes which maydecrease estrogen plasma levels below therapeutic level (for example,anticonvulsant agents; phenytoin, primidone, barbiturates,carbamazepine, ethosuximide, and methosuximide; antituberculous drugssuch as rifampin; antifungal drugs such as griseofulvin). The presentestrogenic substances are less dependent on up- and downregulation ofmicrosomal liver enzymes (e.g. P450's) and also are less sensitive tocompetition with other P450 substrates. Similarly, they do not interferesignificantly in the metabolism of other drugs.

The conjugates of most estrogens, as formed in the liver, are excretedin the bile and may be broken down by gut bacteria in the colon toliberate the active hormone which can then be reabsorbed (enterohepaticrecirculation). There are clinical reports that support the view thatenterohepatic recirculation of estrogens decreases in women takingantibiotics such as ampicillin, tetracycline, etc. Conjugated forms ofthe present estrogenic substances are hardly excreted in the bile,meaning that they are substantially insensitive to drugs that doinfluence the enterohepatic recirculation of other estrogens.

The above observations serve to explain why the estrogenic substances ofthe invention hardly suffer from drug-drug iiiteractions.and thusproduce a very consistent, i.e. predictable, impact. Thus, the efficacyof the estrogenic substances of the invention is highly reliable, whichis particularly important in the field of contraception.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 contains two line-charts showing the competitive displacement of[₃]DT (panel A) and [3H]estradiol (panel B) from the human sexhormone-binding globulin steroid binding site. The unlabeled steroidligands used as competitors were as follows: estetrol (E4), 17α-ethinylestradiol (EE2), 17β-estradiol (E2), testosterone (T) and5α-hihydrotestosterone (DHT).

DETAILED DESCRIPTION OF TIHE INVENTION

Accordingly one aspect of the present invention relates to a method ofcontraception in mammalian females, which method comprises theparenteral or rectal administration of an estrogenic component and aprogestogenic component to a female of childbearing capability in anamount effective to inhibit ovulation, wherein the estrogenic componentis selected from the group consisting of:

substances represented by the following formula

-   -   in which formula R₁, R₂, R₃, R₄ independently are a hydrogen        atom, a hydroxyl group or an alkoxy group with 1-5 carbon atoms;        each of R₅, R₆, R₇ is a hydroxyl group; and    -   no more than 3 of R₁, R₂, R₃, R₄ are hydrogen atoms;

-   precursors capable of liberating a substance according to the    aforementioned formula when used in the present method;

-   and mixtures of one or more of the aforementioned substances and/or    precursors. The term “parenteral administration” as used in here    encompasses transdermal, intranasal, intravaginal, pulmonary,    buccal, subcutaneous, intramuscular and intrauterine administration.

The term “estrogenic component” as used throughout this documentencompasses substances that are capable of triggering an estrogenicresponse in vivo, as well as precursors that are capable of liberatingsuch an estrogenic component in vivo when used in accordance with thepresent invention. In order for estrogenic components to trigger such aresponse they normally have to bind to an estrogen receptor, whichreceptors are found in various tissues within the mammalian body. Theterm “progestogenic component” is defined as a substance that is capableof triggering an progestogenic response in vivo or a precursor which iscapable. of liberating such a substance in vivo. Usually progestogeniccomponents are capable of binding to a progestogen receptor.

It is noted that the present invention not only encompasses the use ofestrogenic and progestogenic components specifically mentioned in thisapplication, but also metabolites of these hormones that displaycomparable in vivo functionality. In this context it is observed that,for instance, levonorgestrel is a metabolite of norgestimate and thatestriol is a metabolite of 17beta-estradiol. Both these progestogens andestrogens have found application in contraceptive formulations and/orhormone replacement therapy. The term “estrogenic substances” as used inthis document does not encompass tritium (³H) labeled estrogenicsubstances such as tritium labeled estetrol.

The present estrogenic substances are distinct from both the biogenicand synthetic estrogens that are commonly applied in pharmaceuticalformulations in that they contain at least 4 hydroxyl groups. Thepresent substances are special in that the 5 membered ring in thesteroid skeleton comprises 3 hydroxyl substituents rather than 0-2.

Known estrogens that contain at least 4-hydroxyl groups and derivativesthereof are:

-   1,3,5(10)-estratrien-2,3,15α,16α,17β-pentol 2-methyl ether-   1,3,5(10)-estratrien-2,3,15β,16α,17β-pentol 2-methyl ether-   1,3,5(10)-estratrien-2,3,16α,17β-tetrol-   1,3,5(10)-estratrien-3,4,16α,17β-tetrol 4-methyl ether-   1,3,5(10)-estratrien-3,15α,16α,17β-tetrol-   1,3,5(10)-estratrien-3,15α,16α,17β-tetrol tetra acetate-   1,3,5(10)-estratrien-3,15β,16β,17β-tetrol tetra acetate

Preferably, the estrogenic substance applied as the active component inthe present composition is a natural estrogen, i.e. an estrogen that isfound in nature and especially in mammals. Even more preferably, theestrogenic substance is a so called biogenic estrogen, i.e. an estrogenthat occurs naturally in the human body, a precursor of a biogenicestrogen or mixtures thereof. Because biogenic estrogens are naturallypresent in the fetal and female body, side-effects are not expected tooccur, particularly not if the serum levels resulting from the exogenousadministration of such estrogens do not substantially exceed naturallyoccurring concentrations. Since estetrol serum levels in the fetus areseveral times higher than those found in pregnant females and knowingthat the fetus is particularly vulnerable, estetrol is deemed to be aparticularly safe biogenic estrogen. Side-effects are not expected tooccur, particularly not if the serum levels resulting from the exogenousadministration of such estrogens do not substantially exceed naturallyoccurring (fetal) concentrations. With synthetic estrogens such asethinyl estradiol there is a (dose dependent) risk of undesirableside-effects, such as thromboembolism, fluid retention, nausea,bloating, cholelithiasis, headache and breast pain.

In a preferred embodiment of the present invention the estrogenicsubstance contains 4 hydroxyl groups. Also, in the aforementionedformula, R₁ preferably represents a hydrogen atom. In said formulapreferably at least 2,more preferably at least 3 of the groups R₁, R₂,R₃ and R₄ represent a hydrogen atom.

The estrogenic substances according to the formula encompass variousenantiomers since the carbon atoms that carry hydroxyl-substituents R₅,R₆ and R₇ are chirally active. In one preferred embodiment, the presentestrogenic substance is 15α-hydroxy substituted. In another preferredembodiment the substance is 16α-hydroxy substituted. In yet another.preferred embodiment, the substances is 17β-hydroxy substituted. Mostpreferably the estrogenic substances are 15α,16α,17β-trihydroxysubstituted.

In another preferred embodiment of the present invention R₃ represents ahydroxyl group or an alkoxy group. In another preferred embodiment thegroups R₁, R₂ and R₄ represent hydrogen atoms, in which case, if R₃, R₅,R₆ and R₇ are hydroxyl groups, the substance is 1,3,5(10)-estratrien-3,15,16,17-tetrol. A preferred isomer of the lattersubstance is 1,3,5 (10)-estratrien-3,15α,16α,17β-tetrol (estetrol).

The invention also encompasses the use of precursors of the estrogenicsubstances that constitute the active component in the present method.These precursors are capable of liberating the aforementioned estrogenicsubstances when used in the present method, e.g. as a result ofmetabolic conversion. These precursors are preferably selected from thegroup of androgenic precursors as well as derivatives of the presentestrogenic substances. Suitable examples of androgenic precursorsinclude androgens that can be converted into the present estrogenicsubstances through iiz vivo aromatisation. Examples of derivatives ofthe present estrogenic substances that can suitably be used asprecursors include such substances wherein the hydrogen atom of at leastone of the hydroxyl groups has been substituted by an acyl radical of ahydrocarbon carboxylic, sulfonic acid or sulfamic acid of 1-25 carbonatoms; tetrahydrofuranyl; tetrahydropyranal; or a straight or branchedchain glycosidic residue containing 1-20 glycosidic units per residue.

Typical examples of precursors which can suitably be used in accordancewith the invention are esters that can be obtained by reacting thehydroxyl groups of the estrogenic substances with substances thatcontain one or more carboxy (M⁺⁻OOC—) groups, wherein M⁺ represents ahydrogen or (akali)metal cation. Hence, in a particularly preferredembodiment, the precursors are derivatives of the estrogenic substances,wherein the hydrogen atom of at least one of the hydroxyl groups in saidformula has been substituted by —CO—R, wherein R is a hydrocarbonradical comprising from 1-25 carbon atoms. Preferably R is hydrogen, oran alkyl, alkenyl or aryl radical comprising from 1-20 carbon atoms.,

The present method usually employs uninterrupted parenteral or rectaladministration of the estrogenic component during a period of at least10 days, preferably of at least 20 days.

The term “uninterrupted” as used in here, means that the estrogeniccomponent is administered at relatively regular intervals, with no(therapeutically) significant interruptions. Naturally, minorinterruptions may occur that do not affect the overall effectiveness ofthe present method, and indeed such aberrations are encompassed by thepresent invention. In a preferred embodiment, and more arithmetically,the administration regimen is deemed to be continuous if the longestinterval between 2 subsequent administrations is not more than 3.5 timesas long as the average interval. Even more preferably said longestinterval is not more than 2.5 times, most preferably not more than 1.5times as long as the average interval.

In the present method, the estrogenic and progestogenic component may beadministered in separate dosage units. However, it is also possible andindeed very convenient to combine these two components into a singledosage unit.

In the contraceptive method according to the present invention thecombination of the progestogenic and estrogenic component is suitablyadministered uninterruptedly during a period of at least 10 days so asto achieve effective ovulation inhibition for a period of at least 20days.

The invention may suitably be reduced to practice in the form of avariety of contraceptive methods that are known to the person skilled inthe art. Amongst these methods are the so called “combined” methods. Thecombined methods make use of monophasic preparations, which containdosage units with a constant amount of an estrogen and a progestogen orbi- or triphasic preparations which have varying levels of estrogen andprogestogen; in most cases consisting of relatively constant levels ofestrogen with a step-wise increase in progestogen throughout the cycle.The combined methods have in common that they are based on a regimenwhich involves an administration-free interval of about 7 days wherebywithdrawal bleeding, simulating the natural menses, occurs. Thus 21 dayintervals of hormone administration alternate with 7 days during whichno hormones are administered.

As an alternative to the aforementioned combined methods, the so called“sequential” method has been proposed. Typical of the sequential methodis that it comprises two consecutive phases, i.e. one phase during whichestrogen and no progestogen is administered and another phase duringwhich a combination of estrogen and progestogen is administered. Thefirst contraceptive sequential methods, like the aforementioned combinedmethods, made use of an administration free interval of about 7 days.More recently, sequential methods have been proposed which do notinclude an administration-free (or placebo) period, meaning thatestrogen is administered throughout the full cycle and that progestogenis co-administered during only part of that cycle. WO 95/17895 (Ehrlichet al.) describes such an uninterrupted sequential method.

Yet another example of a contraceptive method which is encompassed bythe present invention is the so called “continuous combined” method,which is a particular version of the combined method that usesuninterrupted combined administration of a progestogenic and anestrogenic component during a prolonged period of time, e.g. more than50 days. In contrast to ordinary combined and sequential methods, noregular menses occur in the continuous combined method as the continuousadministration of progestogen in the indicated amounts inducesamenorrhoea.

In one embodiment of the invention, which relates to the continuouscombined method, the present method comprises the uninterruptedparenteral or rectal administration of the combination of the estrogeniccomponent and the progestogenic component during a period of at least28,preferably at least 60 days.

In another embodiment of the invention,. which relates to sequential andcombined methods that employ a significant administration-free interval,the method of the invention comprises an interval of at least 2 days,preferably from 3-9 days, most preferably from 5-8 days, during which noprogestogenic component and no estrogenic component is administered andwherein the resulting decrease in serum concentration of theprogestogenic component and the estrogenic component induces menses.

Yet another embodiment of the invention, which concerns a sequentialmethod without a significant pause, is characterised in that itcomprises the uninterrupted parenteral or rectal administration of theestrogenic component during a period of at least 28 days, preferably atleast 60 days, and in that, following the combined administration of theestrogenic component and the progestogenic component, the estrogeniccomponent and no progestogenic component are administered during 3-18consecutive days, preferably during 5-16 consecutive days and theresulting decrease in serum concentration of the progestogenic componentshould normally be sufficient to induce menses.

The mode of administration employed in the present method is suitablyselected from the group consisting of transdermal, intranasal,intravaginal, rectal, pulmonary, buccal, subcutaneous, intramuscular orintrauterine administration. In a particularly preferred embodiment thepresent method employs transdermal, intravaginal, intranasal or rectaladministration. Even more preferably the present method employstransdermal or intranasal administration. The most preferred mode ofadministration is transdermal administration.

Rectal, intranasal, buccal and pulmonary administration are ideallysuited for (at least) once daily administration. Transdermaladministration is advantageously applied at frequencies between once aday and once a month. Intravaginal and intrauterine administrations areadvantageously operated at administration frequencies between onceweekly and once monthly. Subcutaneous and intramuscular administrationare suitably done in the form of depot injections at intervals of 1 weekto 6 months, preferably at intervals of 4 weeks to 3 months.

For reasons of convenience and also to achieve high compliance rates,the present method preferably utilises administration intervals of 1day, 1 week or 1 month. Regimens that employ once daily intranasaladministration, once weekly transdermal administration or once monthlyintravaginal or subcutaneous administration are particularly preferred.

Irrespective of the mode of administration, the estrogenic component ispreferably administered in an amount effective to achieve a blood serumconcentration of at least 1 nanogram per litre, more preferably of atleast 10 nanogram per litre, most preferably at least 100 nanogram perlitre. Generally the resulting blood serum concentration of theestrogenic component will not exceed 100 μg per litre, preferably itwill not exceed 50 μg per litre, more preferably it will not exceed 25μg per litre.

In accordance with the present method the estrogenic component isusually administered in an amount of less than 1 mg per kg of bodyweightper day, preferably of less than 0.4 mg per kg of bodyweight per day,more preferably of less than 0.2 mg per kg of bodyweight per day. Inorder. to achieve a significant impact from the administration of theestrogenic component, it is advisable to administer in an amount of atleast 1 μg per kg of bodyweight per day. Preferably, the administeredamount is at least 2 μg per kg of bodyweight per day, more preferably atleast 5 μg per kg of bodyweight per day. The aforementioned dosages areto be construed as averaged daily dosages in case administrationintervals of more than 1 day are used.

In the present method, particularly when used in humans, the estrogeniccomponent is usually administered parenterally or rectally in an averagedosage of at least 0.05 mg per day, preferably of at least 0.1 mg petday. The maximum parenteral or rectal dosage is normally kept below 40mg per day, preferably below 20 mg per day.

In all of the aforementioned methods it is preferred to parenterally orrectally administer the estrogenic component and the progestogeniccomponent during a period of at least 10,preferably of at least 20 days.In case of a sequential method without pause or a continuous combinedmethod it is preferred to administer the estrogenic component and/or theprogestogenic component uninterruptedly during a period of at least 30days, more preferably of at least 60 days, most preferably of at least150 days. Uninterrupted sequential contraceptive methods, which employcontinuous estrogen administration, exhibit an optimum combination ofcontraceptive reliability and cycle control. The combination of a pauseof 6-7 days during which significant follicular development occurs andthe well documented bad compliance of many pill-users (30%-40% forgetpills occasionally) cause an increased risk of escape ovulationespecially if the pause is (unintentionally) extended. This results in“real life” pregnancy rates of 3-8% per year. By removing the pause andadministering ovulation inhibiting steroids at least once daily, therisk of escape ovulation is much lower.

The general concerns about the so called unopposed administration ofestrogen, i.e. administration of estrogen without co-administeredprogestogen might cause hyperplasia of the endometrium, are lessapplicable to the estrogenic components of the present invention.Therefore, in a particularly preferred embodiment, the presentcontraceptive method is executed in accordance with a sequentialcontraceptive method without pause.

In the present methods the uninterrupted parenteral administration ofthe estrogenic component may usually occur at intervals of at least 12hours, preferably of between 20 hours and 30 days. The relatively highin vivo halflife of the present estrogenic components in comparison tomost known estrogens makes it feasible to employ administrationintervals that are significantly longer than 1 day. With a view tocompliance, however, it is preferred to employ once daily, once weeklyor once monthly administration intervals. Naturally the length of theadministration interval is largely determined by the mode of parenteralor rectal administration that is employed.

In accordance with the present invention the progestogenic component isadvantageously administered in an amount which is effective to achieve ablood serum concentration which is equivalent to at least 50 pg/hllevonorgestrel, preferably of at least 200 pg/mL. In the present method,blood serum concentrations of the progestogenic component will usuallyremain below the equivalent of 10 ng/mL levonorgestrel. Preferably theseconcentrations remain below the equivalent of 2 ng/mL levonorgestrel.

In the present method the progestogenic component is usuallyadministered in an amount of less than 1 mg per kg of bodyweight perday, preferably of less than 0.2 mg per kg of bodyweight per day.Furthermore, it is advisable to parenterally or rectally administer theprogestogenic component in an amount of at least 0. 1 μg per kg ofbodyweight per day. Preferably, the parenterally or rectallyadministered amount is at least 0.3 μg per kg of bodyweight per day.

In human females, the progestogenic component is usually parenterally orrectally administered in an average dosage of at least 5 μg per day,preferably of at least 15 μg per day. The maximum dosages normallyremain below 50 mg per day, preferably below 10 mg per day.

Examples of progestogens which may suitably be used in accordance withthe present invention include: progesterone, levonorgestrel,norgestimate, norethisterone, dydrogesterone, drospirenone,3-beta-hydroxydesogestrel, 3-keto desogestrel (=etonogestrel),17-deacetyl norgestimate, 19-norprogesterone, acetoxypregnenolone,allylestrenol, anagestone, chlormadinone, cyproterone, demegestone,desogestrel, dienogest, dihydrogesterone, dimethisterone, ethisterone,ethynodiol diacetate, flurogestone acetate, gastrinon, gestodene,gestrinone, hydroxymethylprogesterone, hydroxyprogesterbne, lynestrenol(=lynoestrenol), medrogestone, medroxyprogesterone, megestrol,melengestrol, nomegestrol, norethindrone (=norethisterone),norethynodrel, norgestrel (includes d-norgestrel and dl-norgestrel),norgestrienone, normethisterone, progesterone, quingestanol,(17alpha)-17-hydroxy-11-methylene-19-norpregna-4,15-diene-20-yn-3-one,tibolone, trimegestone, algestone acetophenide, nestorone, promegestone,17-hydroxyprogesterone esters, 19-nor-17hydroxyprogesterone,17alpha-ethinyl-testosterone, 17alpha-ethinyl-19-nor-testosterone,d-17beta-acetoxy-13beta-ethyl-17alpha-ethinyl-gon-4-en-3-one oxime andprecursors of these compounds that are capable of liberating theseprogestogens in vivo when used in the present method. Preferably theprogestogen used in the present method is selected from the groupconsisting of progesterone, desogestrel, etonogestrel, gestodene,dienogest, levonorgestrel, norgestimate, norethisterone, drospirenone,trimegestdne, dydrogesterone, precursors of these progestogens andmixtures thereof.

The present method also encompasses the co-administration of activeprinciples in addition to the progestogenic and estrogenic component.For instance, androgens may advantageously be co-administered in orderto prevent symptoms of hypoandrogenicity. Thus, a preferred embodimentof the invention comprises the co-administration of an androgeniccomponent. The androgenic component is suitably co-administered in aneffective amount to suppress symptoms of hypoandrogenicijy.Hypoandrogenicity in females has been associated with mood disturbances,unfavourable changes in haemostatic parameters and lack of bone mass.

The term “androgenic component” is defined as a substance that iscapable of triggering an androgenic response in vivo or a precursorwhich is capable of liberating such a substance in vivo. Usuallyandrogenic components are capable of binding to an androgen receptor.

Androgenic components that may suitably be employed in the presentmethod may be selected from the group consisting of testosterone esterssuch as testosterone undecanoate, testosterone propionate, testosteronephenylpropionate, testosterone isohexanoate, testosterone enantate,testosterone bucanate, testosterone decanoate, testosterone buciclate;testosterone; danazol; gestrinone; methyltestosterone;dehydroepiandrosterone (DHEA); DHEA-sulphate; mesterolon; stanozolol;androstenedione; dihydrotestosterone; androstanediol; metenolon;fluoxymesterone; oxymesterone; methandrostenolol; MENT; precursorscapable of liberating these androgens when used in the present methodand mixtures thereof. Preferably the testosterone esters employed in thepresent method comprise an acyl group which comprises at least 6,morepreferably from. 8-20 and preferably 9-13 carbon atoms. Androgens thatcan be used advantageously in the present method include testosteroneesters,.testosterone and MENT. Most preferably the employed androgen istestosterone undecanoate.

In order to obtain the desired impact from the present method it isadvisable to administer doses in an amount which leads to an increase inblood serum androgen level of at least 0.1 nmole testosterone equivalentper litre, preferably of at least 0.3 nmole testosterone equivalent perlitre. Generally the method leads to an increase in blood serum androgenlevel of no more than 5 nmole testosterone equivalent per litre,preferably of less than 3 nmole testosterone equivalent per litre andmost preferably of less than 1.5 nmole testosterone equivalent perlitre.

The present method preferably does not employ a gonadotropin hormonereleasing hormone composition as described in the aforementioned patentsU.S. Pat. No. 5,211,952,U.S. Pat. No. 5,340,584 and U.S. Pat. No.5,340,585. Similarly, the present method preferably does not employ aluteinizing hormone releasing hormone composition as described in U.S.Pat. No. 4,762,717 and U.S. Pat. No. 5,130,137. Furthermore, the presentmethod preferably does not comprise the co-administration of ananti-progestogen as described in U.S. Pat. No. 5,468,736. The method mayalso suitably be applied without the co-administration of an antisenseoligonucleotide that is complementary to the nucleotide sequence of thefollicle stimulating hormone (FSH) receptor (WO 00/73416).

The present method is not suitable for oophorectomised females or forfemales in whom endometrial stimulation by estrogenic compositions isminimised or absent, e.g. as a result of hysterectomy.

Another aspect of the invention relates to a drug delivery system forparenteral or rectal administration that contain the estrogeniccomponent as defined herein before and a progestogenic component asdescribed herein before, which drug delivery system is selected from thegroup consisting of suppositories, systems for intravaginal delivery,injectable or implantable depot preparations, inhalers, nasal sprays andtransdermal delivery systems, wherein the system contains at least 0.01mg, preferably at least 0.05 mg of the estrogenic component. The systemadditionally contains a progestogenic component, preferably in an amountof at least 10 μg, more preferably it contains at least 30 μg of aprogestogenic component.

In the present kit, the progestogenic component may conveniently becombined with the estrogenic component in a single parenteral or rectaldosage unit, e.g. a single transdermal patch, intravaginal ring,suppository or injection unit.

Transdermal delivery systems include patches, gels, tapes and creams,and can contain excipients such as solubilisers, permeation enhancers(e.g. fatty acids, fatty acid esters, fatty alcohols and amino acids),hydrophilic polymers (e.g. polycarbophil and polyvinyl pyrrolidine) andadhesives and tackifiers (e.g. polyisobutylenes, silicone-basedadhesives, acrylates and polybutene).

Transmucosal delivery systems include patches, suppositories, pessaries,gels, and creams, and can contain excipients such as solubilizers andenhancers (e.g. propylene glycol, bile salts and amino acids), and othervehicles (e.g. polyethylene glycol, fatty acid esters and derivatives,and hydrophilic polymers such as hydroxypropylmethyl cellulose andhyaluronic acid).

Injectable depot systems include solutions, suspensions, gels,microspheres and polymeric injectables, and can comprise excipients suchas solubility-altering agents (e.g. ethanol, propylene glycol andsucrose) and polymers (e.g. polycaprylactones, and PLGA's). Implantabledepot systems include rods and discs, and can contain excipients such asPLGA and polycapryl lactone. Suitable fluid carrier components arephysiologically compatible diluents wherein the active agents can bedissolved, suspended. An example of a diluent is water, with or withoutaddition of electrolyte salts or thickeners. Thus, the depot formulationcan be, for example, an aqueous microcrystalline suspension. Oils areparticularly suitable as diluents, with or without the addition of asolubiliser, of a surfactant, or of a suspension or emulsifying agent.Examples of suitable oils include arachidis oil, olive oil, peanut oil,cottonseed oil, soybean oil, castor oil, and sesame oil. Examples ofsolubilisers include benzyl alcohol and benzyl benzoate. Depotpreparations offer the advantage that a single injection or implantationsuffices for one or several months. Duration of the depot effect dependsthe nature of the estrogenic component (the ester precursors beingpreferred as they display a slower release), the amount of theestrogenic component as well as on the type of carrier substance thatreleases the active agent. Generally, the duration will be in the rangeof 10-30 days, but longer or shorter times can also be achieved.

Other delivery systems that can be used for administering thepharmaceutical composition of the invention include intranasal andpulmonary delivery systems such as sprays and microparticles.

The present invention is further illustrated by the following examples,which, however, are not to be construed as limiting. The featuresdisclosed in the foregoing description, in the following examples and inthe claims may, both separately and in any combination thereof, bematerial for realising the invention in diverse forms thereof.

EXAMPLES Example 1

Vaginal cornification was chosen as a tissue-specific andestrogen-sensitive endpoint to determine the estrogenicity of estetrol(E4), after subcutaneous administration, in hypoestrogenic rats.17β-estradiol (E2) and vehicle (10% ethanol/sesame oil) served ascontrols in the bioassay.

Uterine weight increase in the rat is more commonly used as a measure ofestrogenicity. However, uterine weight also responds to progesterone,testosterone, and other agents not characteristically regarded asestrogens. In the early 1920s it was discovered that follicular fluidfrom the pig ovary contained a factor(s) that causedcornification/keratinization of the vaginal epithelium in the rat (Allenand Doisy, 1923, JAAA, 81, 819-821; Allen and Doisy, 1924,Am. J.Physiol., 69, 577-588). The so-called vaginal cornification response inrats subsequently provided a bioassay for testing estrogenicity. Vaginalepithelial cornification/keratinization in ovariectomized rats can beproduced only by compounds considered to be true estrogens (Jones et al,1973,Fert. Steril. 24, 284-291). Vaginal epithelialcornification/keratinization represents, therefore, a highly selectiveendpoint to determine the potency of estrogens (Reel et al., 1996,Fund.Appli. Toxicol. 34, 288-305).

Adult intact female CD rats were ovariectomized to induce estrogendeficiency. Vaginal lavages were performed daily for seven days toensure that the rats demonstrated castrate vaginal smears (predominanceof leukocytes in the vaginal smear, and similar in appearance to adiestrous vaginal smear). Castrate vaginal smears are indicative thatcomplete ovariectomy was achieved. Treatment commenced followingcompletion of the 7 days of smearing (day 0=first day of dosing).Animals were dosed, once daily for 7 consecutive days. Daily vaginallavages continued to be obtained for 7 days after dosing was initiatedin order to detect vaginal cornification, as an indication of anestrogenic response. A drop of vaginal washings was placed on a glassslide and examined by light microscopy to detect the presence or absenceof cornified epithelial cells. Vaginal lavages were obtained prior todosing on days 0-6 and prior to necropsy on day 7.

The vaginal cornification bioassay was performed in order to determinethe estrogenic profile of E4 when given subcutaneously (sc) toovariectomized adult rats. E2 was used as a positive control. Thevehicle (10% ethanol/sesame oil) served as the negative control.Steroids were dissolved in absolute ethanol and then brought to thefinal concentration with sesame oil (10% ethanol in sesame oil). Theoccurrence of vaginal cornification, indicative of an estrogenicresponse, is an “all or none” response. Data are, therefore, expressedas the number of rats showing a vaginal estrogenic response over thenumber of rats (ratio) treated.

A vaginal estrogenic response occurred in 8/8 rats by day 2 andpersisted through day in rats injected sc with 50 μg/kg/day E2 for 7days (Table 1). Animals treated with the vehicle did not exhibit vaginalepithelial cornification (Table 1). The onset of vaginal epithelialcornification was dose-dependent in rats injected sc with 0.1, 0.3,1.0,and 3.0 mg/kg/day E4 and started at the same day of treatment (Day2) as observed for E2 (Table 1). At 0.1 mg/kg/day E4 already 4/8 ratsand at 0.3 mg/kg/day E4 even 7/8 rats exhibited a vaginal estrogenicresponse by day 7. At 1.0 and 3.0 mg/kg/day E4 all rats showed a vaginalestrogenic response by day 7 (Table 1).

TABLE 1 Vaginal estrogenic response in ovariectomized rats treatedsubcutaneously (sc) with 17β-estradiol (E2) or estetrol (E4). Data areexpressed as the number of rats showing vaginal cornification over thenumber of rats (ratio) treated. Number of Rats Exhibiting EstrogenicResponse/ Number of Rats Treated Treatment Dosing Day of Study Grouproute Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7  0.05 sc 0/8 0/88/8 8/8 8/8 8/8 8/8 8/8 mg/kg/day E2 Vehicle sc 0/8 0/8 0/8 0/8 0/8 0/80/8 0/8 Control 0.1 sc 0/8 0/8 0/8 1/8 1/8 4/8 3/8 4/8 mg/kg/day E4 0.3sc 0/8 0/8 1/8 5/8 7/8 6/8 7/8 7/8 mg/kg/day E4 1.0 sc 0/8 0/8 1/8 6/88/8 7/8 8/8 8/8 mg/kg/day E4 3.0 sc 0/8 0/8 3/8 8/8 8/8 8/8 8/8 8/8mg/kg/day E4

Example 2

To determine the elimination half-life of estetrol (E4) aftersubcutaneous administration (sc), single dose studies were performed infemale Sprague Dawley rats followed by frequent blood sampling over a 24hours interval.

Female Sprague Dawley rats were equipped with a permanent silatic heartcatheter, as described by Kuipers et al. (1985,Gastroenterology, 88,403-411). Rats were allowed to recover from surgery for 5 days and werethan administered 0.05, 0.5,or 5 mg/kg E4 in 0.5 ml arachidis oil. E4was injected in the neck area using a 1 ml syringe and 20 g needle.Blood samples were subsequently collected via the heart catheter inheparinized tubes at 0.5, 1, 2, 4, 8 and 24 hours. Erythrocytes wereremoved by centrifugation at 5000×g for 10 minutes at 4° C. and bloodplasma was stored at −20° C. After thawing the plasma samples,liquid-liquid extraction (hexane and diethyl ether) was employed toprepare the E4-containing plasma samples for HPLC analysis (Perkin Elmer200) and tandem mass spectrometry using a PE Sciex 3000 tandem massspectrometer and APCI interface. With each sample batch, a calibrationcurve with 6 calibrators was recorded. The calibration curve wascalculated using linear regression (correlation coefficient>0.98), whichpermitted quantitation of plasma concentrations. For each rat plasma,sampled at different time intervals, data were collected.

Plasma E4 concentration data were analysed with “WinNonLin, edition 3.1”and involved pharmacokinetic parameters for C_(max), AUC₀₋₂₄ andhalf-life. Interestingly, E4 demonstrated a relatively long half-life of2-3 hours, enabling the detection of bioactive levels of unconjugated E4at all time points over a 24 hour interval.

Example 3

An established competitive steroid-binding assay (Hammond andLahteenmaki. 1983. Clin Chem Acta 132: 101-110) was used to determinethe relative binding affinity of estetrol (E4),17α-ethiinylestradiol(EE2), 17β-estradiol (E2), testosterone (T)and5α-dihydrotestosterone (DHT) for human sex Hormone Binding Globulin(SHBG).

Human SHBG was purified from transgenic mouse serum, as describedpreviously (Avvakumov GV et al., 2000. J Biol Chem 275: 25920-25925).The human SHBG prepared in this way was assessed to be >99% pure bypolyacrylamide gel electrophoresis under denaturing conditions. Itssteroid-binding characteristics are indistinguishable from SHBG in humanserum (Avvakumov GV et al., 2000. J Biol Chem 275: 25920-25925). The invitro assay involved the use of the purified human SHBG and [³H]DHT or[³ H]estradiol as labeled ligands. Human SHBG was treated for 30 min atroom temperature with a dextran-coated charcoal (DCC) suspension inphosphate buffered saline (PBS) to remove any steroid ligand. Aftercentrifugation (2,000×g for 10 min) to sediment the DCC, the supernatantcontaining the human SHBG was diluted in PBS to a concentration of 1 nMbased on its steroid binding capacity.

Duplicate aliquots (100 μl) of this human SHBG solution were thenincubated with an equal volume of either [³H]DHT or [³H]estradiol at 10nM, together with 100 μl of PBS alone or the same amount of PBScontaining increasing concentrations of unlabeled steroid ligands ascompetitors in polystyrene test tubes. After incubation for 1 h at roomtemperature the reaction mixtures were placed in an ice bath for afurther 15 min. Aliquots (600 μl) of an ice cold suspension of DCC werethen added to each tube, and after a brief 2 seconds mixing, each tubewas incubated in an ice bath for either 10 min or 5 min depending onwhether [³H]DHT or [³H]estradiol were being used as labeled ligands,respectively. The unbound ligands adsorbed to DCC were then removed bycentrifugation (2, 000×g for 15 min at 4 C), and the amounts of[³H]labeled ligands bound to SHBG were counted in 2 ml ACS scintillationcocktail using in liquid scintillation spectrophotometer. The averageamounts of [³H]labeled ligands bound to SHBG at each concentration ofcompetitor (B) were expressed as a percentage of the average amounts of[³H]labeled ligands bound to SHBG in the absence of competitor (B₀), andwere. plotted against the concentration of competitor in each assaytube. The results of the competitive binding assays are depicted inFIG. 1. As is clearly apparent from these competitive binding assays,estetrol does not bind at all to human SHBG when tested with either[³H]DHT or [³H]estradiol as labeled ligands. This is in marked contrastwith reference steroids ethinylestradiol, 17β-estradiol, testosteroneand 5α-dihydrotestosterone, which, in this order, show an increasedrelative binding affinity for human SHBG. Importantly, estetrol bindingto SHBG was negligible when compared with the other estrogens tested,ethinylestradiol and 17β-estradiol.

Example 4

A bioassay method is performed to investigate the antiovulatory activityof estetrol (E4), after subcutaneous (sc) administration, in four-daycyclic rats. 17α-ethinylestradiol (EE), 17β-estradiol (E2) and vehicle(10% ethanovsesame oil) serve as controls.

Rats are spontaneously ovulating, polyestrous mammals. Generally,proestrus lasts for 12 to 14 hours, estrus for 25 to 27 hours, metestrusfor 6 to 8 hours, and diestrus for 55 to 57 hours (Freeman, 1988,In: ThePhysiology of Reproduction, E Knobil and J Neill (eds). Raven Press,Ltd, New York, pp. 1893-1928). These stages of the estrous cycle can beclassified based on the cell types present in daily vaginal smears(Schwartz, 1969,Recent Prog. Horm. Res. 25, 1-55).

The preovulatory period of the rat estrous cycle is characterized byovarian follicular growth and enhanced estrogen secretion. In thefour-day cyclic rat, peripheral plasma levels of E2 are basal throughestrus. At the end of metestnis and extending through early diestrus,plasma levels of E2 begin to rise. This increase continues throughdiestrus and early proestrus to reach peak values and plateau bymid-proestrus. Subsequently, E2 levels fall rapidly, reaching basalvalues by the early morning hours of estrus. The rising estrogen levelsfrom late metestrus to early proestrus exert a positive feedback effecton the hypothalamic-pituitary axis resulting in a luteinizing hormone(LH) surge on the afternoon of proestrus. The LH surge inducesfollicular rupture and the release of ova in the early morning hours ofestrus. By early afternoon on the day of estrus, ova are present in theampulla of the oviduct and are readily visualized under a dissectingmicroscope.

Progesterone or levonorgestrel administered subcutaneously on diestrusto four-day is cyclic rats is known to inhibit ovulation and increasedthe estrous cycle length in a dose-dependent manner (Beattie and Corbin,1975,Endocrinology 97, 885-890). It was shown that the progestationalblock of ovulation takes place predominately via thehypothalamic-pituitary axis. Retardation of follicular growthaccompanies ovulatory inhibition at high doses of progestogen when bothserum follicle stimulating hormone (FSH) and LH are significantlyreduced (Beattie and Corbin, 1975,Endocrinology 97, 885-890). De Visseret al. (1984, (Arzneim. Forsch. 34, 1010-1020) have found that oraladministration of EE to rats beginning on the day of estrusand-continuing through the estrous cycle blocked ovulation in adose-dependent manner.

Vaginal smears from female rats are obtained daily for two weeks toselect four-day cycling rats. Only four-day cyclic rats are to be usedfor the antiovulatory bioassay. Starting on the day of estrus, rats aresc dosed, twice daily at 6:30 am and 4:30 pm for 4 consecutive days,with vehicle control (10% ethanol/sesame oil), EE (1.0, 3.0, 30,or 100μg/kg) E2 (0.1, 0.3, 1.0,or 3.0 mg/kg) or E4 (0.1 , 0.3, 1.0,or 3.0mg/kg). One day after the final dose (day 5), rats are euthanized by CO₂asphyxiation at 1 pm, and the number of ova per oviduct is determined.Group means are subsequently calculated for the number of ova perovulated rat (both oviducts). The ratio of rats ovulating for eachtreatment group is compared to the ratio for the vehicle-treated rats.

The results obtained indicate that twice daily sc dosing of EE, E2 andE4 dose-dependently inhibits ovulation, whereas all rats receiving twicedaily vehicle control ovulate. The results of E4 compare favorably to EEand E2. Similar to both EE and E2,complete ovulation inhibition isachieved with higher dosages of E4. Furthermore, E4's antiovulatoryactivity is of the same order of magnitude as E2,showing equal potencyor even more potency in inhibiting ovulation than E2.

Example 5

Suitable formulations for the transdermal administration of estrogensare known in the art, and may be employed in the methods of the presentinvention. For example, suitable transdermal patch formulations for theadministration of exogenous estrogen are described in U.S. Pat. No.4,460,372 (Campbell et al.), U.S. Pat. No. 4,573,996 (Kwiatek et al.),U.S. Pat. No. 4,624,665 (Nuwayser), U.S. Pat. No. 4,722,941 (Eckert etal.), U.S. Pat. No. 5,223,261 (Nelson et al.), the disclosures of whichare hereby incorporated by reference.

One suitable type of transdermal patch for use in the methods of thepresent invention includes a backing layer which is non-permeable, apermeable surface layer, an adhesive layer substantially continuouslycoating the permeable surface layer, and a reservoir located orsandwiched between the backing layer and the permeable surface layersuch that the backing layer extends around the sides of the reservoirand is joined to the permeable surface layer at the edges of thepermeable surface layer. The reservoir contains the estrogenic componentand is in fluid contact with the permeable surface layer. Thetransdermal patch is adhered to the skin by the adhesive layer on thepermeable surface layer, such that the permeable surface layer is insubstantially continuous contact with the skin when the transdermalpatch is adhered to the skin.

While the transdermal patch is adhered to the skin of the subject, theestrogenic component contained in the reservoir of the transdermal patchis transferred via the permeable surface layer, through the adhesivelayer, and to and through the skin of the subject. The transdernal patchmay suitably include one or more penetration-enhancing agents in thereservoir that enhance the penetration of the estrogenic componentthrough the skin.

Examples of suitable materials which may comprise the backing layer arewell known in the art of transdermal patch delivery, and anyconventional backing layer material may be employed in the transdermalpatch of the instant invention. Specific examples of suitable backinglayer materials include but are not limited to polyester film, such ashigh density polyethylene, low density polyethylene or composites ofpolyethylene; polypropylene; polyvinyl chloride, polyvinylidenechloride; ethylene-vinyl acetate copolymers; and the like.

Examples of suitable permeable surface layer materials are also wellknown in the art of transdermal patch delivery, and any conventionalmaterial which is permeable to the estrogenic component, maybe employedin the transdermal patch of the instant invention. Specific examples ofsuitable materials for the permeable surface layer include but are notlimited to dense or microporous polymer films such as those comprised ofpolycarbonates, polyvinyl chlorides, polyamides, modacrylic copolymers,polysulfones, halogenated polymers, polychloroethers, acetal polymers,acrylic resins, and the like. Specific examples of these types ofconventional permeable membrane materials are described in U.S. Pat. No.3,797,494 to Zaffaroni.

Examples of suitable adhesives which may be coated on the backing layerto provide the adhesive layer are also well known in the art andinclude, for example pressure sensitive adhesives such as thosecomprising acrylic and/or methacrylic polymers. Specific examples ofsuitable adhesives include polymers of esters of acrylic or methacrylicacid (e.g., n-butanol, n-pentanol, isopentanol, 2-methyl butanol,1-methyl butanol, 1 -methyl pentanol, 3-methyl pentanol, 3-methylpentanol, 3-ethyl butanol, isooctanol, n-decanol, or n-dodecanol estersthereof) alone or copolymerized with ethylenically unsaturated monomerssuch as acrylic acid, methacrylic acid, acrylamnide, methacrylamide,N-alkoxymethyl acrylamides, N-alkoxymethyl methacrylamides,N-t-butylacrylamide, itaconic acid, vinyl acetate, N-branched C.sub.10-24 alkyl maleamic acids, glycol diacrylate, or mixtures of theforegoing; natural or synthetic rubbers such as silicon rubber,styrene-butadiene rubber, butyl-ether rubber, neoprene rubber, nitrilerubber, polyisobutylene, polybutadiene, and polyisoprene; polyurethaneelastomers; vinyl polymers such as polyvinyl alcohol, polyvinyl ethers,polyvinyl pyrrolidone, and polyvinyl acetate; ureaformraldehyde resins;phenol formaldehyde resins; resorcinol formaldehyde resins; cellulosederivatives such as ethyl cellulose, methyl cellulose, nitrocellulose,cellulose acetatebutyrate, and carboxymethyl cellulose; and natural gumssuch as guar, acacia, pectin, starch, destria, gelatin, casein, etc.

As will be apparent to those skilled in the art, the adhesive layershould be inert to the estrogenic component, and should not interferewith the transderrmal delivery of the estrogenic component through thepermeable surface layer. Pressure sensitive adhesives are preferred forthe adhesive layer of the transdermal patch to facilitate theapplication of the patch to the skin of the subject.

Suitable penetration-enhancing agents are well known in the art as well.Examples of conventional penetration-enhancing agents include alkanolssuch as ethanol, hexanol, cyclohexanol, and the like; hydrocarbons suchas hexane, cyclohexane, isopropylbenzene; aldehydes and ketones such ascyclohexanone, acetamide; N,N-di(oower alkyl)acetamides such asN,N-diethylacetamide, N,N-dimethyl acetamide,;N-(2-hydroxyethyl)acetarnide; esters such as N,N-di-ower alkylsulfoxides; essential oils such as propylene glycol, glycerine, glycerolmonolaurate, isopropyl myristate, and ethyl oleate; salicylates; andmixtures of any of the above.

In another example of a transdermal patch which is suitable for thetransdermal delivery of the estrogenic component according to thepresent invention, said estrogenic component is incorporated into theadhesive layer rather than being contained in a reservoir. Examples ofthese types of patches are conventionally known and include, forexample, the CLIMERA.®. patch available from Berlex. This type oftransdermal patch comprises a backing layer and an adhesive/drug layer.The adhesive/drug layer has. the combined function of adhering the patchto the skin of the subject and containing the estrogenic component,which is to be administered. The active ingredient is leached from theadhesive/drug layer to and through the skin of the subject when thepatch is adhered to the skin.

Any of the backing layers described herein above may be employed in thisembodiment as well. In addition, any of the suitable adhesives describedabove may be employed. The adhesive/drug layer comprises a relativelyhomogeneous mixture of the selected adhesive and the active ingredient.Typically, the adhesive/drug layer comprises a coating substantiallycovering one surface of the backing layer. The adhesive/drug layer mayalso include a penetration enhancing agent such as those described aboveby incorporating the penetration enhancing agent into the substantiallyhomogeneous mixture of the adhesive and the active ingredient.

As will be readily apparent to those skilled in the art, the transdermalpatches according to the present invention may include a variety ofadditional excipients which are conventionally employed to facilitatethe transdermal administration of the estrogenic component. Examples ofsuch excipients include but are not limited to carriers, gelling agents,suspending agents, dispersing agents, preservatives, stabilisers,wetting agents, emulsifiing agents, and the like. Specific examples ofeach of these types of excipients are well known in the art and anyconventional excipients may be employed in the transdermal patches ofthe instant invention.

The amount of estrogenic component contained in the transdermal patchformulations will depend upon the precise form of estrogenic componentto be administered, but should be sufficient to deliver at least 20 μgper day. The amount of progestogenic component to be administered istypically equivalent to an amount of at least 20 μg levonorgestel perday. Typically, the transdermal patches are designed to be worn forseveral days before replacement is required. Thus the amount ofestrogenic component in the patch must be sufficient to permit theadministration of at least 20 μg per day for a period of several days.As an example, a transdermal patch according to the present inventionwhich is designed to administer around 400 μg of estetrol and 20 μglevonorgestel per day for seven (7) days would contain approximately 40mg of the estrogen and approximately 2 mg of the progestogen. Based uponthis information, one skilled in the art would be able to establish thenecessary amount of estrogenic component to be included in a giventransdermal patch to achieve the delivery of the correct daily dose ofestrogenic component.

Example 6

Suitable nontoxic pharmaceutically acceptable carriers for use in a drugdelivery system for intranasal administration of the present estogeniccomponent will be apparent to those skilled in the art of nasalpharmaceutical formulations. For those not skilled in the art, referenceis made to “Remington's Pharmaceutical Sciences”, 4th edition, 1970.Obviously, the choice of suitable carriers will depend on the exactnature of the particular nasal dosage form desired, e.g. whether theestrogenic component is to be formulated into a nasal solution (for useas drops or as a spray), nasal microspheres, a nasal suspension, a nasalointment or a nasal gel, as well as on the identity of the estrogeniccomponent.

Examples of the preparation of typical nasal compositions are set forthbelow.

Nasal Solution:

-   -   15 mg of estetrol and 15 mg of progesterone are combined with 10        mg of Tween 80. That mixture is then combined with a quantity of        isotonic saline sufficient to bring the total volume to 50 ml.        The solution is sterilised by being passed through a 0.2 micron        Millipore filter.        Nasal Gel:    -   250 ml of isotonic saline are heated to 80° C. and 1.5 g of        Methocel are added, with stirring. The resultant mixture is        allowed to stand at room temperature for 2 hours. Then, 25 mg of        estetrol and 25 mg of progesterone are mixed together with 10 mg        of Tween 80. The estetrdl/Tween mixture and a quantity of        isotonic saline sufficient to bring the total volume to 500 ml        were added to the gel and thoroughly mixed.

Examnple 7

The intravaginal drug delivery vehicle may suitably take the form of avaginal ring. Vaginal rings are torous shaped devices designed todeliver a relatively constant dose of drug to the vagina usually over aperiod of weeks to months. Typically, they are made of a poly. EVAelastomer and the estrogenic component is released by diffusion thoughthe elastomer. The vaginal ring is designed to regulate the release rateof the estrogenic component so as to provide the user with theappropriate daily dose. Among the important factors governing releaseare the solubility of the estrogenic component in the ring elastomer,the surface area of the drug reservoir, the distance the drug mustdiffuse through the ring body to reach its surface and the molecularweight of the drug.

If relatively high release rates are desired, they can be attained by adrug load at the ring surface as is characteristic of the homogeneousmatrix ring design. This design, however, suffers from rapidly decliningrelease rates as the distance the drug must travel to reach the ringsurface increases as the drug load near the surface is depleted. Ifmoderately high release rates are needed to provide the appropriatedose, a design which modulates release rate by imposing a layer ofdrug-free elastomer between the drug reservoir and the ring exterior isappropriate. This may be attained by coating a homogeneous ring, or toconserve drug, by incorporating a drug-free core, a shell design may beused. If an even lower release rate is desired, the drug may be confinedto a small diameter at the center of the ring (“core ring”). Numeroustypes of vaginal rings have been described in the patent and non-patentliterature alike.

An example of the preparation of an estetrol containing intravaginalring is set forth below:

Four 58 nmm core rings are prepared as follows. Fifty grams of Silastic382® are mixed with 0.3 g of stannous octoate, transferred to a 50 ccplastic syringe and injected into four brass ring moulds. After 45minutes, the moulds are opened, the rings removed, the flash is trimmedand the rings are cut open at a 45° angle. A mixture of 84.4 g Silastic382®, 24.2 g of micronised estetrol and 12.2 g micronised levonorgestrelare mixed in a Teflon bowl. The mixture is transferred to a Lucitecoating cup with a bottom opening of 8.7 mm. The open rings are heatedat 110° C. for 30 minutes, cooled and weighed. The open rings weighapproximately 9.8 g. The open rings are pulled through the coating cupand dipped in a solution of 0.67% stannous octoate in toluene (w/v). Theopen ring is again heated at 110° C. for 30 minutes and reweighed. Theweight of the coated open ring is approximately 10.3 g and the weight ofthe coating on the open rings is therefore approximately 0.5 g.

In order to apply the outer layer a 16.5 cm long piece of siliconerubber tubing having 6.3 mm diameter and 0.3 mm wall thickness isswollen in hexane and the open ring coated with the medicated layer isplaced inside the silicone rubber tubing. The hexane is evaporated atroom temperature and the tubing contracted to the size of the open ringforming an outer layer having a thickness of 0.2 mm.

The excess tubing is trimmed flush with the ends of the open ring andDow Corning Medical Adhesive A is applied at both ends of the open ringand to 1 cm of the outer layer at both ends of the open ring. A 4 cmpiece of silicone tubing 6.3 mm inner diameter and 0.3 mm wall thicknessis swollen with hexane and placed over the two ends of the open ring toclose the ring. The ring is held for about two minutes until the tubinghas shrunk and fits snugly over the ring junction. The adhesive isallowed to cure for 24 hours, the rings are rinsed in alcohol and airdried.

Example 8

An estetrol containing depot formulation can suitably be prepared as setforth below.

At room temperature, 1000 mg estetrol and 1500 mg levonorgestrel aredispersed in 6 millilitre dehydrated ethanol. This solution is thendiluted with 660 ml arachidis oil under thorough stirring. The resultingsolution is sterilised by filtration.

In case an estetrol ester is used, e.g. estetrol valerate esters, asignificantly lower release rate can be obtained. Such low release ratesare particularly advantage if the depot injections are to beadministered at relatively long time intervals, e.g. intervals of morethan 1 week.

1. A drug delivery system for parenteral, or rectal administration,comprising an estrogenic component and a progestogenic component, saiddrug delivery system being selected from the group consisting ofsuppositories, systems for intravaginal delivery, injectable orimplantable depot preparations, inhalers, nasal sprays and transdermaldelivery systems, wherein the system contains at least 0.01 mg of theestrogenic component, wherein said estrogenic component is selected fromthe group consisting of substances represented by the following formula:

in which formula R₁, R₂, R₃, R₄, independently are a hydrogen atom, ahydroxyl group or an alkoxy group with 1-5 carbon atoms; each of R₅, R₆,R₇ is a hydroxyl group; and no more than 3 of R₁, R₂, R₃, R₄ arehydrogen atoms; precursors capable of liberating a substance accordingto the aforementioned formula when used in the present method, whichprecursors are derivatives of the substances represented by the formula,wherein the hydrogen atom of at least one of the hydroxyl groups in saidformula has been substituted by an acyl radical of a hydrocarboncarboxylic, sulfonic or sulfamic acid of 1-25 carbon atoms;tetrahydrofuranyl; tetrahydropyranal; or a straight or branched chainglycosidic residue containing 1-20 glycosidic units per residue; andmixtures of one or more of the aforementioned substances and/orprecursors, and wherein the system does not contain a luteinizinghormone releasing hormone (LHRH) composition or a gonadotropin hormonereleasing hormone (GnRH) composition.
 2. The drug delivery systemaccording to claim 1, wherein the device contains at least 10 μg of theprogestogenic component.